Approximately 20% of the US population suffers from non-alcoholic fatty liver disease (NAFLD) that can progress to hepatosteatosis, fibrosis, cirrhosis and hepatocellular carcinoma. Hepatic secretion of VLDL plays an essential role in regulating intrahepatic and intravascular lipid homeostasis. The overproduction of hepatic VLDL characterizes the pathogenesis of hyperlipidemia in obesity and diabetes and contributes significantly to an increased risk of cardiovascular disease. Despite the significant impact of abnormalities in VLDL secretion on human health, fundamental cellular mechanisms that drive VLDL assembly are yet to be understood. Hepatic VLDL assembly and secretion is significantly reduced by protein misfolding in the endoplasmic reticulum (ER) and increased by the adaptive unfolded protein response (UPR). The proposed research will elucidate the mechanistic role of the UPR in maintaining triglyceride (TG) levels in the ER lumen for VLDL assembly. We will vigorously test the hypothesis that changes in the ER protein-folding environment of the hepatocyte activate inositol-requiring kinase1? (IRE1?) to splice Xbp1 mRNA to generate XBP1s in response to a nutritional stimulus are key in the etiology of NAFLD and abnormal lipid secretion and hyperlipidemia. Our hypothesis is based on three recent fundamental findings regarding assembly of VLDL. First, we have demonstrated that nutritional excess alters lipid homeostasis and activates the UPR in hepatocytes. Aim 1 will uncover the mechanism that links metabolic stress with protein misfolding in the ER. Aims 2 and 3 focus on how XBP1s is essential role to maintain ER lipid homeostasis. We have demonstrated that XBP1s provides two independent functions for assembly of TG-rich VLDL. First, XBP1s activates transcription of protein disulfide isomerase (Pdi1) to maintain microsomal TG transfer protein (MTP) in an active form to promote lipid assembly with apolipoprotein B (ApoB). Aim 2 will study how nutritional stress reduces MTP activity in a PDI1-dependent manner. Aim 3 will identify the second mechanism by which XBP1s is essential to promote TG-rich VLDL assembly in an MTP-independent manner using a combination of mRNA-Seq and mass spectrometry approaches. Finally, and significantly, our data show that PDI1 promotes oxidative folding of ApoB, thus for the first time uncovering a protein folding substrate for PDI1 in vivo. Aim 4 will use hepatocyte-specific Pdi1-null mice to study how PDI1 promotes ApoB100 oxidative folding and alters interactions with the protein-folding environment (i.e., molecular chaperones). Results from the proposed studies will elucidate the mechanisms by which IRE1?/XBP1 maintains hepatocyte lipid homeostasis by partitioning TG into the ER lumen for VLDL assembly. The findings will provide fundamental understanding of mechanisms that govern VLDL maturation, which will facilitate the development of novel therapeutic approaches to treat hepatosteatosis and hyperlipidemia.